The eukaryotic heat shock protein 90s (Hsp90s) are ubiquitous chaperone proteins, which bind and hydrolyze ATP. The Hsp90 family of proteins includes four known members: Hsp90-alpha and -beta, Grp94, and Trap-1. The roles of Hsp90s in cellular functions are not completely understood, but recent studies indicate that Hsp90s regulate client proteins involved in cellular signaling (Wegele et al., 2004, Rev Physiol Biochem Pharmocol 151:1-44). These client proteins include key proteins involved in signal transduction, cell cycle control, and transcriptional regulation (Burrows et al., 2004, Cell Cycle, 3:e20-e26; Pratt et al., 2004, Cellular Signalling, 16:857-872). For example, researchers have reported that Hsp90 chaperone proteins are associated with important signaling proteins, such as steroid hormone receptors and protein kinases, including many implicated in tumorigenesis, such as Raf-1, EGFR, v-Src family kinases, Cdk4, and ErbB-2 (Buchner, 1999, TIBS, 24:136-141; Stepanova et al., 1996, Genes Dev., 10:1491-1502; Dai et al., 1996, J. Biol. Chem., 271:22030-22034).
In vivo and in vitro studies indicate that without the aid of co-chaperones, Hsp90is unable to fold or activate proteins. For steroid receptor conformation and association in vitro, Hsp90 requires Hsp70 and p60/Hop/Sti1 (Caplan, 1999, Trends in Cell Biol., 9:262-268). In vivo Hsp90 may interact with Hsp70and its co-chaperones. Other co-chaperones associated with Hsp90s in higher eukaryotes include Hip, Hsp40/Hdj2/Hsj1, Immunophilins, p23, and p50 (Caplan, 1999, Trends in Cell. Biol., 9:262-268). The activation of Hsp90requires both the association with co-chaperones and ATP hydrolysis by Hsp90. Inhibition of Hsp90ATPase activity has proven an effective means to disrupt Hsp90 activity and consequently the many signaling pathways that it regulates including cell cycle pathways required for tumor growth (Kamal et., 2004, Trends in Mol. Med., 10:283-290). Inhibition of Hsp90activity has become an important target in the development of effective cancer therapy (Maloney and Workman, 2002, Expert Opin. Biol. Ther., 2:3-24; Neckers, 2003, Cur. Med. Chem. 10:733-739). While the ATPase activity of Hsp90 is the most studied target, the chaperone has many other vulnerable points (Chiosis et al., 2004, Drug Discovery Today, 9:881-888).
Ansamycin antibiotics are natural products derived from Streptomyces hygroscopicus that have profound effects on eukaryotic cells. Many ansamycins, such as herbimycin A (HA) and geldanamycin (GM), bind tightly to a pocket in the HSP90 protein (Stebbins et al., 1997, Cell, 89:239-250). The binding of ansamycins to Hsp90 has been reported to inhibit protein refolding and to cause the proteasome dependent degradation of a select group of cellular proteins (Sepp-Lorenzino et al., 1995, J. Biol. Chem., 270:16580-16587; Whitesell et al., 1994, Proc. Natl. Acad. Sci. USA, 91:8324-8328).
The ansamycins were originally isolated on the basis of their ability to revert v-src transformed fibroblasts (Uehara et al., 1985, Jpn. J. Cancer Res., 76:672-675). Subsequently, they were said to have antiproliferative effects on cells transformed with a number of oncogenes, particularly those encoding tyrosine kinases (Uehara et al., 1988, Virology, 164:294-298). Inhibition of cell growth is associated with apoptosis and, in certain cellular systems, with induction of differentiation (Vasilevskaya et al., 1999, Cancer Res., 59:3935-3940).
The use of ansamycins as anticancer agents are described in U.S. Pat. Nos. 4,261,989, 5,387,584, and 5,932,566. The preparation of the ansamycin, geldanamycin, is described in U.S. Pat. No. 3,595,955.
The ansarnycin-binding pocket in the N-terminus of Hsp90 is highly conserved and has weak homology to the ATP-binding site of DNA gyrase (Stebbins et al., 1997, Cell, 89:239-250; Grenert et al., 1997, J. Biol. Chem., 272:23843-23850). This pocket has been reported to bind ATP and ADP with low affinity and to have weak ATPase activity (Prodromou et al., 1997, Cell, 90:65-75; Panaretou et al., 1998, EMBO J., 17:4829-4836). In vitro and in vivo studies are said to indicate that occupancy of the pocket by ansamycins alters Hsp90 function and inhibits protein refolding. At high concentrations, ansamycins have been reported to prevent binding of protein substrates to Hsp90 (Scheibel et al., 1999, Proc. Natl. Acad. Sci. USA, 96:1297-1302; Schulte et al., 1995, J. Biol. Chem., 270:24585-24588; Whitesell et al., 1994, Proc. Natl. Acad. Sci. USA, 91:8324-8328). Alternatively, they have also been reported to inhibit the ATP-dependent release of chaperone-associated protein substrates (Schneider et al., 1996, Proc. Nati. Acad. Sci. USA, 93:14536-14541; Sepp-Lorenzinoet al., 1995, J. Biol. Chem., 270:16580-16587). In both models, the unfolded substrates are said to be degraded by a ubiquitin-dependent process in the proteasome (Scheibel et al., 1999, Proc. Nati. Acad. Sci. USA, 96:1297-1302; and Sepp-Lorenzino et al., 1995, J. Biol. Chem., 270:16580-16587).
In both tumor and nontransformed cells, binding of ansamycins to Hsp90 has been reported to result in the degradation of a subset of signaling regulators. These include Raf (Schulte et al., 1997, Biochem. Biophys. Res. Commun., 239:655-659; Schulte et al., 1995, J. Biol. Chem., 270:24585-24588), nuclear steroid receptors (Segnitz, 1997, J. Biol. Chem., 272:18694-18701; Smith et al., 1995, Mol. Cell. Biol., 15:6804-6812), v-src (Whitesell et al., 1994, Proc. Natl. Acad. Sci. USA, 91:8324-8328) and certain transmembrane tyrosine kinases (Sepp-Lorenzino et al., 1995, J. Biol. Chem., 270:16580-16587) such as EGF receptor (EGFR) and Her2/Neu (Hartmann et al., 1997, Int. J. Cancer, 70:221-229; Miller et al., 1994, Cancer Res., 54:2724-2730; Mimnaugh et al., 1996, J. Biol. Chem., 271:22796-22801; Schnur et al., 1995, J. Med. Chem., 38:3806-3812). The ansamycin-induced loss of these proteins is said to lead to the selective disruption of certain regulatory pathways and results in growth arrest at specific phases of the cell cycle (Muise-Heimericks et al., 1998, J. Biol. Chem., 273:29864-29872).
Geldanamycin is a benzoquinone ansamycin polyketide isolated from Streptomyces hygroscopicus var. geldanus. Although originally discovered by screening microbial extracts for antibacterial and antiviral activity, geldanamycin was later found to be cytotoxic to certain tumor cells in vitro and to reverse the morphology of cells transformed by the Rous sarcoma virus to a normal state.
Geldanamycin's nanomolar potency and apparent specificity for aberrant protein kinase dependent tumor cells, as well as the discovery that its primary target in mammalian cells is the ubiquitous Hsp90 protein chaperone, have stimulated interest in the development of this anti-cancer drug. However, the association of hepatotoxicity with the administration of geldanamycin led to its withdrawal from Phase I clinical trials. As with several other promising anticancer agents, geldanamycin also has poor water solubility that makes it difficult to deliver in effective doses.
More recently, attention has focused on 17-amino derivatives of geldanamycin, in particular 17-(allylamino)-17-desmethoxygeldanamycin (17-AAG), that show reduced hepatotoxicity while maintaining Hsp90 binding. Certain 17-amino derivatives of geldanamycin, 11-oxogeldanamycin, and 5,6-dihydrogeldanamycin, are disclosed in U.S. Pat. Nos. 4,261,989, 5,387,584, and 5,932,566. Like geldanamycin, 17-AAG has limited aqueous solubility.
Treatment of cancer cells with geldanamycin or 17-AAG causes a retinoblastoma protein-dependent G1 block, mediated by down-regulation of the induction pathways for cyclin D-cyclin dependent cdk4 and cdk6 protein kinase activity. Cell cycle arrest is followed by differentiation and apoptosis. G1 progression is unaffected by geldanamycin or 17-AAG in cells with mutated retinoblastoma protein; these cells undergo cell cycle arrest after mitosis, again followed by apoptosis.
The mechanism of action of benzoquinone ansamycins appears to be via binding to Hsp90 and subsequent degradation of Hsp90-associated client proteins. Among the most sensitive client protein targets of the benzoquinone ansamycins are the Her kinases (also known as ErbB), Raf, Met tyrosine kinase, and the steroid receptors. Hsp90 is also involved in the cellular response to stress, including heat, radiation, and toxins. Certain benzoquinone ansamycins, such as 17-AAG, have thus been studied to determine their interaction with cytotoxins that do not target Hsp90 client proteins.
U.S. Pat. Nos. 6,245,759, 6,306,874, and 6,313,138, disclose compositions comprising certain tyrosine kinase inhibitors together with 17-AAG and methods for treating cancer with such compositions. Munster, 2001, Clinical Cancer Research, 7:2228-2236, discloses that 17-AAG sensitizes cells in culture to the cytotoxic effects of paclitaxel and doxorubicin, and that the sensitization towards paclitaxel by 17-AAG is schedule-dependent in retinoblastoma protein-producing cells due to the action of these two drugs at different stages of the cell cycle. Treatment of cells with a combination of paclitaxel and 17-AAG is reported to give synergistic apoptosis, while pretreatment of cells with 17-AAG followed by treatment with paclitaxel is reported to result in abrogation of apoptosis. Treatment of cells with paclitaxel followed by treatment with 17-AAG 4 hours later is reported to show a synergistic effect similar to coincident treatment. Citri et al., 2002, EMBO Journal, 21:2407-2417, discloses an additive effect upon co-administration of geldanamycin and an irreversible protein kinase inhibitor, CI-1033, on growth of ErbB2-expressing cancer cells in vitro.
As can be seen from this discussion, inhibitors of the ATPase activity of Hsp90, such as geldanamycin and radicicol, have significant activity and a broad range as anti-tumor drugs. Considerable resources are being directed to develop derivatives of these drugs that are stable and do not have objectionable side effects. Some of these drugs are currently in clinical trials. The development of Hsp90 ATPase inhibitors without significant side effects is an important goal of cutting edge cancer research.
Thus, what is needed is the discovery of additional agents that interact with Hsp90 and that can be used in the treatment of conditions, particularly various cancers or other cell proliferative disorders, as well as viral infections.